Extracellular vesicles derived from horse mesenchymal stromal cells: microRNA profiling in the context of equine osteoarthritis

Osteoarthritis (OA) represents the most common cause of retirement from athletic activities in horses [1]. Although current pharmacological treatments can be used to alleviate symptoms, the restoration of damaged cartilage is not achieved [2]. Therefore, mesenchymal stromal cells (MSCs) have emerged as a promising alternative in regenerative medicine owing to their paracrine effects, largely mediated by extracellular vesicles (EVs), used as key means of communication between cells. A relevant EVs’ cargo is represented by microRNAs (miRNAs), significant post-transcriptional gene regulators whose gene expression can be modulated by the surrounding extracellular microenvironment [3]. As further research is needed to explore their role in EVs and how MSC preconditioning influences them, the aim of this study was to characterize EVs secreted by horse MSCs, investigating miRNA expression changes under different extracellular conditions mimicking equine OA environment. In this study, primary horse MSCs derived from bone marrow and fat tissue collected from carcasses of slaughtered horses, were used to produce pools of 3 individuals for each tissue. Three adipose tissue-derived MSC pools were cultured under standard conditions and exposed to pro-inflammatory stimuli (IL-1β and TNF-α). EVs were isolated via ultracentrifugation and characterized for size distribution and concentration through Nanoparticle Tracking Analysis. In parallel, a literature analysis was performed to identify a panel of miRNAs linked to the inflammatory response of OA. MiRNAs were therefore extracted from isolated EVs and amplified through RT-qPCR, and the expression of miRNAs selected from literature was evaluated. MSC-derived EVs were successfully isolated, with a concentration mean of 2.57 x 1010 particles/mL and a size mean of 139.40 nm. Extracted miRNAs showed a concentration ranging from 0.705 to 4.48 ng/μL. Nine out of the 23 miRNAs linked to OA from literature were detected through RTqPCR in untreated MSCs. A preliminary assessment of these selected miRNAs was carried out on the 3 adipose-derived MSC pools to determine whether exposure to pro-inflammatory cytokines affects EVs content. These preliminary results confirm the presence of miRNAs associated with the inflammatory process of OA within extracellular vesicles derived from equine MSCs. Expanding the analyses by increasing sample size will allow to understand how EV miRNAs are differentially expressed according to changes in MSCs milieu.